Chronic lymphocytic leukemia (CLL) is heterogeneous clinically, reflecting functional alterations in multiple pathways, including apoptotic and B cell signaling. Novel targeted agents in late-stage development include PI3K and pro-apoptotic B cell leukemia/lymphoma 2 (BCL-2) homology 3 (BH3) mimetics. We have a long- standing interest in understanding the fundamental mechanisms for the response and resistance to BH3 mimetics and active agents, such as fludarabine. We have identified several determinants with biologic and clinical significance in the apoptotic response and resistance to these therapeutics, with the BCL-2 family proteins emerging as key regulators of cellular survival. We will address two critical therapy resistance mechanisms: impaired apoptosis and autophagy, processes in which the anti-apoptotic BCL-2 family is crucial, as supported by our published and preliminary data. We have developed an apoptotic index based on BCL-2 family gene expression that defines sensitivity to ABT-737 in CLL and have generated and characterized ABT- 737 and ABT-199-resistant cell lines. Increased MCL-1 expression in resistant cells was critical to preventing cell death, with the excess MCL-1 being associated with the BH3-only protein, BIM, or key autophagy regulators Beclin 1 and Beclin 2, which also contain a BH3 domain. ABT-199-resistant cells also developed high BCL-xL expression that is regulated epigenetically through miR377. Both miR377 and BCL-xL expression predicted clinical outcome in CLL and thus can provide new targets for intervention as well as biomarkers for therapy. Therapy-resistant cells were addicted to high basal autophagy that depended on Beclin 2, instead of Beclin 1, and AMPK, and surprisingly, also had high mTORC1 activity. Our central hypothesis is that responsiveness and resistance to CLL therapeutics are governed by the BCL-2 family proteins that regulate apoptosis and autophagy. We will perform experiments with leukemia cell lines and primary CLL patient cells after clinical therapy with ABT-199, cultured ex vivo in suspension or on stromal cells to mimic their interaction with their in vivo microenvironment. Our specific aims are: 1) To define the role of BCL-xL and MCL-1 in resistance to and sensitization to BH3 mimetics, 2) To define autophagy as a mechanism of resistance, and 3) To lay the foundations for optimizing the use of BCL-2 targeting agents by integrating gene expression data obtained from cell lines with that from patients treated in the clinic or ex vivo. Our likelihood of success is enhanced by the expertise of the PI in the BCL-2 family biology, apoptosis, and autophagy, M. Smith, B. Hill (Co-I) in CLL and lymphoid malignancies, E. Hsi (Co-I) in pathology, and A. Ting (Consultant) in methylation and next-generation sequencing. Understanding the critical BCL-2 family protein associations according to differing cell sensitivities will allow development of molecular and pharmacologic approaches for their effective targeting. By characterizing apoptotic and autophagy pathways in lymphoid malignancies, we seek to ultimately use them to predict response and thus personalize the selection of targeted therapeutic agents.